All Herpes viruses assemble their DNA-packaged capsids in the infected cell nucleus. These capsids then undergo a process unique in biology: they bud into the inner nuclear membrane and pinch off into the perinuclear space to form a primary enveloped particle. These particles subsequently fuse their envelopes with the outer nuclear membrane and the now cytoplasmic capsids undergo a secondary envelopment in the cytoplasm to form the mature virion. At each envelopment step a complex of proteins termed the tegument is assembled between the capsid and envelope and likely drives the envelopment process. The primary enveloped and mature HSV particles are morphologically quite distinct and are believed to contain quite different arrays of tegument and envelope proteins. However the structure and composition of the perinuclear HSV virus (pnHSV) is poorly defined because to date it has only been possible to examine it by ultrastructural studies. In this application I describe methodology that we have developed to purify pnHSV particles in biochemically useful quantities. We propose to extend our ongoing studies by performing a proteomic analysis of the pnHSV and to test the role of cellular proteins which we have already shown to be present. Furthermore, using the model tegument protein vhs we will dissect the molecular mechanism by which a tegument polypeptide is targeted into the enveloping viral particle. PUBLIC HEALTH RELEVANCE: Herpes simplex viruses (HSV) types 1 and 2 are best known for their role in the generation of oral and genital mucocutaneous lesions, however HSV infection of the central nervous system can also lead to fatal encephalitis, and replication in the eye is a leading cause of blindness in the United States each year. Correct assembly of the HSV particle is critical for the production of an infectious virion, and the spread of disease. This application investigates HSV assembly at the molecular level and will have great therapeutic significance, since it is expected to yield a large and diverse array of new targets for anti-HSV drugs.